JP2024507379A - Artificial TRAP cage, its use, and method of its preparation - Google Patents

Abstract

The present invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by a cross-linking agent, the cross-linking agents being selected for their specific characteristics, whereby the cage is An artificial TRAP cage is provided that is programmable to open or remain closed on demand under specific conditions.

Description

The present invention belongs to the field of biochemistry. The present invention is an engineered protein cage, termed a "TRAP cage," containing a selected number of TRAP rings held in place by a molecular cross-linking agent, the cross-linking agent selected for their specific characteristics. relates to an artificial protein cage, whereby the cage is programmable to open or remain closed on demand under specific conditions.

Protein complexes in nature are important and highly precise biological nanomachines and nanostructures. Large protein complexes in nature are typically assembled from several individual proteins held together by non-covalent interactions (ie, hydrogen bonds, hydrophobic packing). This is particularly evident in protein cages such as capsids, where multiple copies of the same protein subunit are thus kept together. In synthetic structural biology, it can be useful to be able to design and construct artificial protein associations, potentially allowing the introduction of properties and abilities that do not occur in nature. Toward this goal, new schemes that connect individual proteins together in a defined manner are desirable.

Recently, we have investigated such a possibility using TRAP (trp RNA binding attenuation protein) from Geobacillus stearothermophilus as a nanometer building block. This TRAP, in its native state and along with several other ring proteins, adopts an 11-subunit oligomeric ring structure and has proven to be a useful bionano building block.

Given the disadvantages of known processes, the inventors are attempting to find other ways to connect protein subunits. Although there are some disclosures regarding linking two or other numbers of proteins via their cysteine SH groups, we have taken into account the use of gold as a "stitching" reagent in this field. focused on.

The use of gold compounds to incorporate gold particles into nanostructures or as targeting molecules in delivery systems, providing nanoparticles as nanoclusters, protein cages for multiple applications, among others, is well documented in the literature. as well as in patent documents, which are prior art to the present invention. For example, International Application No. PCT/KR2013/004454 surface-modifies gold nanoparticles with hyaluronic acid, which has biocompatible, biodegradable, and liver tissue-specific delivery properties, and has excellent stability in the body; We present a method for preparing hyaluronic acid-gold nanoparticle/protein complexes that can be used as liver-targeted drug delivery systems by attaching protein drugs to the unmodified surface of gold nanoparticles to treat liver diseases. Describe it.

US patent application no. discloses the introduction of noble metal atoms, such as gold, into cage-like proteins such as.

International Application No. PCT/US2011/034190 discloses that two or more nanoparticles (gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or Disclosed is an antibody-nanoparticle conjugate comprising an antibody-nanoparticle conjugate (such as an alloy of two or more thereof).

Another example is US Patent Application No. US 14/849,379, which discloses a recombinant self-associating protein comprising a target-directing peptide fused to the self-associated protein and a self-associated gold ion reducing peptide.

International Application No. PCT/IB2018/056150 discloses a gold-donor agent in which an -S-Au-S- bond is formed, characterized in that the gold-donor agent is a halogen (triarylphosphine) gold(I). Disclosed are methods for the conjugation of free thiol groups containing biomolecules leading to biomolecular complex formation, including reactions that connect the biomolecules using donor agents. Furthermore, the use of halogen (triarylphosphine) gold(I) molecules as gold-donor agents in methods of biomolecular complex formation is disclosed therein.

A. D. Malay et al.: “An ultra-stable gold-coordinated protein cage displaying reversible assembly”; Nature 569 (2019): 438-442 Publication (herein incorporated by reference). ) between the thiol groups of the cysteine pair We disclose TRAP cages bound together by a single gold atom, ie, an Au(I) ion, which formed a linear coordinate bond to the Au(I) ion.

In the present invention, the TRAP rings forming the artificial TRAP cage are held in place by crosslinkers that are not made from metal atoms, and the crosslinkers are selected for their specific characteristics, whereby the cage is A new approach is realized, not through -S-Au-S- bonds, which are programmable to open or remain closed on demand under specific conditions. This approach is revolutionary from a state-of-the-art perspective, allowing control of the assembly and disassembly of capsid-like protein complexes. A new approach regarding metal ions as cross-linking agents is also demonstrated, which is innovative from a state-of-the-art perspective and also allows control of the assembly and disassembly of capsid-like protein complexes.

The subject of the first aspect of the invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by a molecular cross-linking agent, the cross-linking agent a selected molecule and not a single atom, e.g. a metal atom, whereby the cage is programmable to open or remain closed as required under specific conditions; It is an artificial TRAP cage.

Preferably, said specific conditions correspond to a specific cleavage characteristic of the molecular cross-linking agent.

The use of molecular crosslinkers rather than single atom crosslinkers provides a greater degree of design choice and flexibility in designing the cage. These allow for increased programmability and control of crosslinker cleavage characteristics. There is a much wider range of molecular crosslinkers available for selection than (metallic) atomic crosslinkers.

All known TRAP cage syntheses prior to this have utilized atomic gold or mercury crosslinkers, and no work has contemplated the use of molecular crosslinkers. Since all previous teachings focused on gold atoms, the larger size of the molecules and the different chemistries required of them to cross-link the TRAP rings meant that the molecular cross-linkers This means that it is not clear that it could perform a crosslinking function leading to cage formation.

Preferably, the specific cleavage characteristics of the molecular cross-linking agent are
(i) reduction-resistant/insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions;
(ii) a reduction-responsive/sensitive molecular cross-linker, whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular cross-linker, whereby the cage opens upon exposure to light. selected from the group.

Preferably, the reduction resistant/insensitive molecular crosslinker may be selected from the group comprising bismaleimideohexane (BMH), bisbromobimane and bis-bromoxylene.

Preferably, the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising dithiobismaleimideoethane (DTME).

Preferably, the photoactivatable molecular crosslinkers include bis-halomethylbenzene, as well as 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m -BBN), and derivatives thereof, including 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

Preferably, the molecular crosslinkers are homobifunctional molecular moieties and derivatives thereof.

Preferably, the homobifunctional molecular crosslinker is bismaleimidohexane (BMH).

Preferably the cage is resistant/insensitive to reducing conditions.

Preferably, the homobifunctional molecular crosslinker is dithiobismaleimidoethane (DTME).

Preferably the cage is responsive/sensitive to reducing conditions.

Preferably, the molecular crosslinker is bis-halomethylbenzene and its derivatives.

Preferably, the molecular crosslinking agent is from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB). selected from.

Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

Preferably, the number of TRAP rings in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35. preferably 12 to 34 pieces, preferably 13 to 33 pieces, preferably 14 to 32 pieces, preferably 15 to 31 pieces, preferably 16 to 30 pieces, preferably 17 to 29 pieces, preferably 18 to 28 pieces , preferably 19 to 27 pieces, preferably 20 to 26 pieces. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP rings in the TRAP cage is 12-24.

Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

Preferably, the cage according to the invention comprises a mixture of different programmable molecular crosslinkers.

Preferably, the cage according to the invention encapsulates said cargo, which can be programmed to deliver said cargo at specific times and at desired locations.

Preferably, the artificial TRAP cage protein is modified to include any one or more mutations selected from the group comprising K35C, R64S, and K35C/R64S. Preferably, the engineered TRAP cage protein is modified to include the K35C mutation. Preferably, the cross-linking agent comprises dithiobismaleimideethane (DTME) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably, the cage according to the invention is hollow. Preferably, the cage according to the invention is approximately spherical in shape, preferably hollow spherical. A cage herein has a hollow shape roughly approximating a hollow sphere. These are close to the shapes obtained if the TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then connected together. For any shape, the vertices and edges are imaginary, i.e. there is no real physical polyhedron on which the TRAP ring is placed, rather the shape of the TRAP cage is as if the ring were placed on the vertex or corner of a regular convex polyhedron, and then It looks like they are connected together.

Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is raised above normal room temperature or human/animal body temperature, preferably between 0 and 100°C, preferably between 15 Stable at ~100°C, preferably stable at 15-75°C, preferably up to 75°C, preferably below 75°C.

Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable between pH 3 and 11, preferably stable between pH 4 and 10. and is preferably stable at pH 5 to 9.

Preferably, the TRAP cage contains a chaotropic agent (an agent that will disrupt hydrogen bonds, disrupt or denature protein or macromolecular structure in solution), or that will disrupt or denature protein or macromolecular structure. Stable in surfactants that would otherwise be expected. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable up to at least 7M urea. Preferably, the TRAP cage is stable at up to 15% SDS. The stability of the cages described herein can be tested using these agents under standard conditions that would be known to those skilled in the art to demonstrate said stability.

The cage described herein exhibits unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

The subject of the invention is also the use of the cage according to the invention as defined above in the delivery of cargo in a controlled period and to a desired location.

The subject matter of the present invention also relates to the use of any one or more of homobifunctional molecular moieties and groups comprising bis-halomethylbenzene and its derivatives as programmable cross-linking agents in the construction of programmable TRAP cages. be.

The subject of a second aspect of the invention is a method of preparing an artificial TRAP cage, the method comprising:
(i) obtaining the TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system;
(ii) conjugation of the TRAP ring unit via at least one free thiol linkage with a programmable molecular crosslinker, where the crosslinker is selected for its specific characteristics;
(iii) formation of a TRAP cage; and (iv) purification and isolation of a TRAP cage.

Preferably, step (ii) first comprises conjugation of the TRAP ring unit via at least one metal crosslinker, preferably an atomic metal crosslinker. Step (ii) then comprises replacing the metal crosslinker with a molecular crosslinker. Molecular crosslinkers can exchange metal atoms without changing the orientation of the rings in the cage. Preferably the metal is gold. This modified step (ii) is preferably applied when the crosslinking agent is a photocleavable linker, preferably the crosslinking agent is bromoxylene or bisbromobimane.

Preferably, the programmable crosslinker is
(i) a reduction-resistant/insensitive linker, whereby the cage remains closed under reducing conditions;
(ii) a reduction-responsive/sensitive linker, whereby the cage opens under reducing conditions; and (iii) a light-activatable linker, whereby the cage opens upon exposure to light. Ru.

Preferably, the reduction resistant/insensitive molecular crosslinking agent may be selected from the group comprising bismaleimidohexane (BMH) and bis-bromoxylene.

Preferably, the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising dithiobismaleimidoethane (DTME).

Preferably, the photoactivatable molecular crosslinkers include bis-halomethylbenzene, as well as 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m -BBN), and derivatives thereof, including 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB). Preferably, the artificial TRAP cage protein is modified to include any one or more mutations selected from the group comprising K35C, R64S, and K35C/R64S. Preferably, the engineered TRAP cage protein is modified to include the K35C mutation.

Preferably, the cross-linking agent comprises dithiobismaleimideethane (DTME) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably, the number of TRAP rings in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35. preferably 12 to 34 pieces, preferably 13 to 33 pieces, preferably 14 to 32 pieces, preferably 15 to 31 pieces, preferably 16 to 30 pieces, preferably 17 to 29 pieces, preferably 18 to 28 pieces , preferably 19 to 27 pieces, preferably 20 to 26 pieces. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP rings in the TRAP cage is 12-24.

Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

Preferably, step (ii) involves conjugation with a mixture of different programmable crosslinkers.

Preferably, the reduction resistant/insensitive molecular crosslinking agent may be selected from the group comprising bismaleimidohexane (BMH) and bis-bromoxylene.

Preferably, the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising dithiobismaleimidoethane (DTME).

Preferably, the photoactivatable molecular crosslinkers include bis-halomethylbenzene, as well as 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m -BBN), and derivatives thereof, including 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

If cysteines are not present in the biomolecule, or if they are present but not available for reaction, -SH groups, preferably as radicals of cysteine, can be introduced into the biomolecule.

Introduction of cysteine can be performed by any method known in the art. For example, cysteine introduction can be performed by methods known in the art, including, but not limited to, commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers. do not have. The methods described above are known by those skilled in the art and ready-to-use kits with protocols are commercially available.

The -SH moiety can also be introduced into the biomolecule by modification of other amino acids in the biomolecule, ie by site-directed mutagenesis or by solid phase peptide synthesis.

The subject of the invention is also the TRAP cage produced by the method. These cages may have any of the features or characteristics described in connection with the first aspect of the invention above, or any others described herein.

The subject of a third aspect of the invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one crosslinking agent comprising a metal. Preferably, the crosslinking agent contains only metals. Preferably, the metal is a metal ion, preferably of a single type of metal.

Preferably, the metal crosslinker is selected for specific characteristics, whereby the cage is programmable to open or remain closed as required under said specific conditions.

Preferably the metal is selected from the group comprising Ag(I), Cd(II), Zn(II) and Co(II). Metals can be derivatives of these metals.

Preferably the metal is a d10 metal with a non-linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Zn(II) or Co(II).

Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Ag(I) or Cd(II).

Preferably, the artificial TRAP cage proteins are K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/ modified to include any one or more mutations selected from the group comprising K35C.

Preferably, the artificial TRAP cage protein is modified to include a K35C/S33H mutation or a K35H/S33H mutation.

Preferably, the cross-linking agent comprises silver (Ag(I)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cadmium (Cd(II)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cobalt (Co(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably, the cross-linking agent comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably, the number of TRAP rings in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35. preferably 12 to 34 pieces, preferably 13 to 33 pieces, preferably 14 to 32 pieces, preferably 15 to 31 pieces, preferably 16 to 30 pieces, preferably 17 to 29 pieces, preferably 18 to 28 pieces , preferably 19 to 27 pieces, preferably 20 to 26 pieces. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP rings in the TRAP cage is 12-24.

Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

Preferably, the cage according to the invention contains a mixture of different crosslinking agents.

Preferably, the cage according to the invention encapsulates said cargo, which can be programmed to deliver said cargo at specific times and at desired locations.

Preferably, the cage according to the invention is hollow. Preferably, the cage according to the invention is approximately spherical in shape, preferably a hollow sphere. A cage herein has a hollow shape roughly approximating a hollow sphere. These are close to the shapes obtained if the TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then connected together.

Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is raised above normal room temperature or human/animal body temperature, preferably between 0 and 100°C, preferably between 15 Stable at ~100°C, preferably stable at 15-75°C, preferably up to 75°C, preferably below 75°C.

Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable between pH 3 and 11, preferably stable between pH 4 and 10. and is preferably stable at pH 5 to 9.

Preferably, the TRAP cage contains a chaotropic agent (an agent that will disrupt hydrogen bonds, disrupt or denature protein or macromolecular structure in solution), or that will disrupt or denature protein or macromolecular structure. Stable in surfactants that would otherwise be expected. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable up to at least 7M urea. Preferably, the TRAP cage is stable at up to 15% SDS. The stability of the cages described herein can be tested using these agents under standard conditions that would be known to those skilled in the art to demonstrate said stability.

The cage described herein exhibits unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

The subject of the invention is also the use of the cage according to the invention as defined above in the delivery of cargo in a controlled period and to a desired location.

The subject of the invention is the use of the metals Ag(I), Cd(II), Zn(II) and Co(II), and any one or more of their derivatives, as cross-linking agents in the construction of TRAP cages. Also of multiple use.

The subject of a fourth aspect of the invention is a method of preparing an artificial TRAP cage, the method comprising:
(i) obtaining the TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system;
(ii) conjugation of the TRAP ring unit via at least one free thiol linkage with a metal crosslinker, where the crosslinker is selected for its specific characteristics;
(iii) formation of a TRAP cage; and (iv) purification and isolation of a TRAP cage.

Preferably the metal is selected from the group comprising Ag(I), Cd(II), Zn(II) and Co(II). Metals can be derivatives of these metals.

Preferably the metal is a d10 metal with a non-linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Zn(II) or Co(II).

Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Ag(I) or Cd(II).

Preferably, the artificial TRAP cage proteins are K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/ modified to include any one or more mutations selected from the group comprising K35C.

Preferably, the artificial TRAP cage protein is modified to include a K35C/S33H mutation or a K35H/S33H mutation.

Preferably, the cross-linking agent comprises silver (Ag(I)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cadmium (Cd(II)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cobalt (Co(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably, the cross-linking agent comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably, the number of TRAP rings in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35. preferably 12 to 34 pieces, preferably 13 to 33 pieces, preferably 14 to 32 pieces, preferably 15 to 31 pieces, preferably 16 to 30 pieces, preferably 17 to 29 pieces, preferably 18 to 28 pieces , preferably 19 to 27 pieces, preferably 20 to 26 pieces. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP rings in the TRAP cage is 12-24.

Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

If cysteines are not present in the biomolecule, or if they are present but not available for reaction, -SH groups, preferably as radicals of cysteine, can be introduced into the biomolecule.

Introduction of cysteine can be performed by any method known in the art. For example, cysteine introduction can be performed by methods known in the art, including, but not limited to, commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers. do not have. The methods described above are known by those skilled in the art and ready-to-use kits with protocols are commercially available.

The -SH moiety can also be introduced into the biomolecule by modification of other amino acids in the biomolecule, ie by site-directed mutagenesis or by solid phase peptide synthesis.

Preferably, the method is carried out partially or completely in HEPES buffer. Preferably, the method is carried out at about pH8.

The subject of the invention is also the TRAP cage produced by the method. These cages may have any of the features or characteristics described in connection with the first aspect of the invention above, or any others described herein.

A subject of the invention is also the use of any of the TRAP cages described herein as a medicament.

A subject of the invention is also a method of treating a patient, comprising administering to said patient a TRAP cage as described herein.

A subject of the invention is also the use of any of the TRAP cages described herein in treating diseases in a patient.

The subject of the invention is also the TRAP cage produced by the method. These cages may have any of the features or characteristics described above in connection with the previous aspects of the invention, or any others described herein.

The subject of a further aspect of the invention is an artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one crosslinking agent. These cages may have any of the features or characteristics described above in connection with the previous aspects of the invention, or any others described herein.

Preferably the crosslinking agent is metal. Preferably, the crosslinking agent contains only metals. Preferably, the metal is a metal ion, preferably of a single type of metal.

Preferably, the metal crosslinker is selected for specific characteristics, whereby the cage is programmable to open or remain closed as required under said specific conditions.

Preferably the metal is selected from the group comprising Au(I), Ag(I), Cd(II), Zn(II) and Co(II). Metals can be derivatives of these metals.

Preferably the metal is a d10 metal with a non-linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Zn(II) or Co(II).

Preferably, the metal is a d10 metal with a two-ligand linear coordination structure or shell. Preferably, the d10 metal with a nonlinear coordination structure or shell is Ag(I) or Cd(II).

Preferably, the artificial TRAP cage proteins are K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/ modified to include any one or more mutations selected from the group comprising K35C.

Preferably, the artificial TRAP cage protein is modified to include a K35C/S33H mutation or a K35H/S33H mutation.

Preferably, the cross-linking agent comprises silver (Ag(I)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cadmium (Cd(II)) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably the cross-linking agent comprises cobalt (Co(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably, the cross-linking agent comprises zinc (Zn(II)) and preferably the artificial TRAP cage protein is modified to contain the S33H/K35C or S33H/K35H mutations.

Preferably the cross-linking agent comprises gold (Au(I)) and preferably the artificial TRAP cage protein is modified to contain S33C/R64S.

Preferably, the specific cleavage characteristics of the molecular cross-linker include (i) a reduction-resistant/insensitive molecular cross-linker, whereby the cage remains closed under reducing conditions; (ii) a reduction-responsive/sensitive molecule; (iii) a photoactivatable molecular cross-linker, whereby the cage opens upon exposure to light;

Preferably, the reduction resistant/insensitive molecular crosslinker may be selected from the group comprising bismaleimidohexane (BMH), bisbromobimane, and bis-bromoxylene.

Preferably, the reduction responsive/sensitive molecular crosslinker may be selected from the group comprising dithiobismaleimidoethane (DTME).

Preferably, the photoactivatable molecular crosslinkers include bis-halomethylbenzene, as well as 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m -BBN), and derivatives thereof, including 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

Preferably, the molecular crosslinkers are homobifunctional molecular moieties and derivatives thereof. Preferably, the homobifunctional molecular crosslinker is bismaleimidohexane (BMH).

Preferably the cage is resistant/insensitive to reducing conditions. Preferably, the homobifunctional molecular crosslinker is dithiobismaleimidoethane (DTME).

Preferably the cage is responsive/sensitive to reducing conditions. Preferably, the molecular crosslinker is bis-halomethylbenzene and its derivatives.

Preferably, the molecular crosslinking agent is from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB). selected from.

Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

Preferably, the artificial TRAP cage protein is modified to include any one or more mutations selected from the group comprising K35C, R64S, and K35C/R64S. Preferably, the engineered TRAP cage protein is modified to include the K35C mutation.

Preferably, the cross-linking agent comprises dithiobismaleimideethane (DTME) and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation.

Preferably, the number of TRAP rings in the TRAP cage is between 6 and 60, preferably between 7 and 55, preferably between 8 and 50, preferably between 9 and 45, preferably between 10 and 40, preferably between 11 and 35. preferably 12 to 34 pieces, preferably 13 to 33 pieces, preferably 14 to 32 pieces, preferably 15 to 31 pieces, preferably 16 to 30 pieces, preferably 17 to 29 pieces, preferably 18 to 28 pieces , preferably 19 to 27 pieces, preferably 20 to 26 pieces. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is greater than 6, preferably greater than 10, preferably greater than 15, preferably greater than 20.

Preferably, the number of TRAP rings in the TRAP cage is 12-24.

Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

Preferably, the cage according to the invention contains a mixture of different crosslinking agents.

Preferably, a cage according to the invention encapsulates cargo, which may be delivered at a specific time and at a desired location.

Preferably, the cage according to the invention is hollow. Preferably, the cage according to the invention is approximately spherical in shape, preferably a hollow sphere. A cage herein has a hollow shape roughly approximating a hollow sphere. These are close to the shapes obtained if the TRAP rings were placed at the vertices or corners of a regular convex polyhedron and then connected together.

Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is raised above normal room temperature or human/animal body temperature, preferably between 0 and 100°C, preferably between 15 Stable at ~100°C, preferably stable at 15-75°C, preferably up to 75°C, preferably below 75°C.

Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable between pH 3 and 11, preferably stable between pH 4 and 10. and is preferably stable at pH 5 to 9.

Preferably, the TRAP cage contains a chaotropic agent (an agent that will disrupt hydrogen bonds, disrupt or denature protein or macromolecular structure in solution), or that will disrupt or denature protein or macromolecular structure. Stable in surfactants that would otherwise be expected. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable up to at least 7M urea. Preferably, the TRAP cage is stable at up to 15% SDS. The stability of the cages described herein can be tested using these agents under standard conditions that would be known to those skilled in the art to demonstrate said stability.

The cage described herein exhibits unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

The subject of the invention is also the use of the cage according to the invention as defined above in the delivery of cargo in a controlled period and to a desired location.

The subject matter of the present invention includes K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/K35C It is also a TRAP cage that includes a protein that has been modified to include any one or more of the mutations selected from the group.

The subject matter of the present invention includes K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/K35C It is also a TRAP protein modified to include any one or more mutations selected from the group.

Reference herein to "TRAP protein" refers to the bacterial protein Tryptophan RNA Binding Attenuation Protein. The protein can be isolated, for example, from wild type Geobacillus stearothermophilus or other such bacteria. Although this protein can be isolated from a variety of bacteria, TRAP proteins that may function as described herein include Alkalihalobacillus ligniniphilus, Anaerobacillus isosaccharinicus, Anoxybacillus caldiproteoli ticus, Anoxybacillus calidus, Anoxybacillus pushchinoensis, Anoxybacillus tepidamans, Anoxybacillus tepidamans, Anoxybacillus vitaminophilus, Bacillaceae bacteria, Bacillus alveayuensis, Bacillus alveayuensis, Bacillus sinesaloumensis, B acillus sp. FJAT-14578, Bacillus sp. HD4P25, Bacillus sp. HMF5848, Bacillus sp. PS06, Bacillus sp. REN16, Bacillus sp. SA1-12, Bacillus sp. V3-13 , Bacillus timonensis, Bacillus timonensis, Bacillus weihaiensis, Bacillus yapensis, Calidifontibacillus erzurumensis, Calidifo ntibacillus oryziterrae, Cytobacillus luteolus, Fredinandcohnia aciditolerans, Fredinandcohnia humi, Fredinandcohnia onubensis, Fredinandcohnia onubensis, Geobacillus genomosp. 3, Geobacillus sp. 46C-IIa, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus thermodenitrificans NG80-2, Halo bacillus dabanensis, Halobacillus halophilus, Halobacillus halophilus, Jeotgalibacillus proteolyticus, Litchfieldia alkalitelluri s, Litchfieldia salsa, Mesobacillus harenae, Metabacillus, Metabacillus litoralis, Metabacillus sediminilitoris, Oceanobacillus limi, Oceanobacillus species Castelsardo, Ornithinibacillus, Ornithinibacillus b variensis, Ornithinibacillus contaminans, Ornithinibacillus halophilus, Ornithinibacillus scapharcae, Parageobacillus caldoxylos Parageobacillus thermoglucosidas, Parageobacillus thermoglucosidas, Parageobacillus thermoglucosidas ius, Parageobacillus thermoglucosidasius, Paucisalibacillus globulus, Paucisalibacillus spp. EB02, Priestia abyssalis, Priestia endophytica, Priestia filamentosa, Priestia koreensis, Priestia megaterium, Psychrobacillus gl Thermolongibacillus altinsuensis.

The Trp RNA binding attenuation protein is a bacterial cyclic homo-11-mer (A. A. Antson, J. Otridge, A. M. Brzozowski, E. J. Dodson, G. A. (See G. Dodson, K. S. Wilson, T. Smith, M. Yang, T. Kurecki, P. Gollnick). The structure of the trp RNA binding attenuation protein can be found in the literature (Nature 374, 693-700 (1995), herein incorporated by reference).

Suitably, the protein used herein is a modified version of wild type TRAP isolated from Bacillus stearothermophilus. This can be seen in Table 1:

The wild type TRAP Bacillus stearothermophilus gene sequence is found in Table 2:

Preferably, protein preparation is carried out by biomolecule expression in a suitable expression system and purification of the expression product. Preferably, a modified version of the wild-type TRAP Bacillus stearothermophilus gene sequence described above is used.

TRAP proteins form a ring, herein a "TRAP ring", which is the natural state of the TRAP protein. Typically, as in the case of the Geobacillus Stearothermophilus proteins demonstrated herein, TRAP monomeric proteins spontaneously associate into cyclic bodies or rings made from monomers.

TRAP cages are only formed under certain conditions, such as those demonstrated herein, with the presence of cysteines that can be crosslinked, resulting in rings associated with the cage. For example, as demonstrated herein, these will be formed with the presence of cysteine at position 35 (a result of the K35C mutation).

Reference herein to a "TRAP ring" is synonymous with a TRAP building block, a subunit of a TRAP cage complex, or a TRAP monomer association. Reference herein to "analogs" of a particular protein or nucleotide sequence means that it is capable of performing the specified function, such as TRAP cage formation under the conditions described herein, or that the specified function is Refers to a protein or nucleotide sequence that has sufficient identity or homology with the protein or nucleotide sequence to encode a protein capable of TRAP cage formation, eg, under the conditions described herein.

To determine the percent identity/homology of two sequences, the sequences in question and a reference are aligned for optimal comparison purposes (e.g., the first and second amino acids or Gaps may be introduced in one or both of the nucleic acid sequences and non-homologous sequences may be ignored for comparison purposes). The sequence preferably comprises at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 75% of the amino acids or nucleotides of the reference sequence of relevant length. , 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100%, it can be determined to be an analog of a specific thing. When comparing amino acid residues or nucleotides at corresponding amino acid or nucleotide positions, if a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecule are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap.

Suitably, the TRAP protein has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% identity or homology with the amino acid sequence of SEQ ID NO: 1 Contains amino acid sequence. Preferably, the TRAP protein comprises an amino acid sequence that has at least 85% identity or homology to the amino acid sequence of SEQ ID NO:1.

Reference herein to a "TRAP cage" refers to an associated protein complex formed from multiple biomolecules, herein multiple TRAP protein rings forming a complex. TRAP protein rings can be linked together by a crosslinker, herein a molecular crosslinker. "Complex," "association," and "aggregate" are used alternatively in this description to refer to a superstructure constructed by reactions between biomolecules. The amount of units involved in the complex depends on the nature of the biomolecule. More specifically, it resides in the amount of biomolecules and the amount of --SH groups present on the biomolecules. "TRAP cage" and "artificial TRAP cage" are used interchangeably herein.

TRAP proteins are suitable biomolecular models for the methods of the invention. This is due to its high intrinsic stability, toroidal shape, lack of natural cysteine residues (for easier control of the conjugation process), as well as the fact that the resulting cysteine is well suited for proper bond formation. This is probably due to the availability of residues that can be converted to cysteine in a unique chemical and spatial environment.

Reference herein to "programmable" means that the TRAP cages of the present invention tend to behave in a specific and selected manner upon exposure to specific environmental conditions or stimuli or It is intended to convey that they have properties imparted or manipulated that make them susceptible to or predisposed to behave.

Reference herein to "open" is synonymous with a TRAP cage that has shattered, leaked, fragmented, broken, or generally allowed cargo to escape from the interior of the cage.

Reference herein to "closed" is synonymous with a TRAP cage that remains intact, unbreakable, impervious, or generally the entire cage.

Reference herein to "bifunctional" refers to a molecule having two functional groups, e.g. a molecule having two functional groups herein, for connecting the TRAP rings in the TRAP cage. There is one functional group for each cysteine thiol group to be crosslinked. Reference herein to "homobifunctional" refers to a bifunctional linker in which the two groups are the same.

Preferably, homobifunctional linkers include bismaleimidehexane (BMH), dithiobismaleimidoethane (DTME), bis-halomethylbenzene and its derivatives, 2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromo xylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

"Unit", "subunit", "molecule", "biomolecule", "monomer" are used alternatively in this description, and refer to one molecule that connects to another molecule to form a complex. means.

"Molecular crosslinker" means a molecule that acts to connect a unit, subunit, molecule, biomolecule, or monomer to other instances thereof through the formation of one or more chemical bonds. It is. Molecular crosslinkers are not single atom linkers that are separate entities.

References herein to "reduction-resistant/insensitive molecular cross-linkers" are references to cross-linkers that are not cleaved by reductive reactions, such as those typically found when disulfide bonds are cleaved by reducing agents. be. These crosslinkers are stable under conditions that would result in cleavage of reduction-sensitive bonds. These include bismaleimidohexane (BMH) and bis-bromoxylene.

References herein to "reduction-responsive/sensitive molecular cross-linkers" are references to cross-linkers that are cleaved by reductive reactions, such as those typically found when disulfide bonds are cleaved by reducing agents. be. These crosslinkers are not stable under conditions that would result in cleavage of reduction sensitive bonds. These include dithiobismaleimidoethane (DTME).

Reference herein to a "photoactivatable molecular crosslinker" is a reference to a crosslinker that is photoreactive or sensitive to light, i.e., one that will be cleaved when exposed to light. be. This light may be UV or other such light in a specific range of wavelengths. These are 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro- Contains benzene (BDNB).

Protein cages in which the building blocks are held together by simple cross-linkers through facile chemistries that allow for easy interchangeability to achieve programmable rather than easily inducible disassembly. has been proposed (Fig. 1a). Disassembly of such cages will depend on the cleavage characteristics of the crosslinker employed. To test this possibility, TRAP (K35C/R64S) has been used, using either the simple bifunctional molecular cross-linkers dithiobismaleimideethane (DTME) or bismaleimidehexane (BMH). Attempts have been made to connect TRAP. They acted as connectors like Au(I) in the gold-mediated association (TRAP cage ^Au(I) ) thanks to the thiol-specific reaction at neutral pH. Because DTME contains scissile disulfide bonds, whereas BMH does not, the two resulting cages (TRAP-caged ^DTME and TRAP-caged ^BMH ) exhibit contrasting disassembly characteristics when exposed to reducing agents. (Fig. 1a).

To obtain a covalently cross-linked cage, TRAP (K35C/R64S) was mixed with either DTME or BMH in an aqueous buffer. Size exclusion chromatography (SEC) of the resulting reaction mixture showed a substantial peak at an elution volume similar to that of the TRAP cage ^Au(I) , suggesting successful cage formation with both crosslinkers. (Figure 1). The isolated fractions were further analyzed by SEC (Fig. 1c), dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM) (Fig. 1d, Fig. 2), showing that approximately 25 nm in diameter This yielded the expected results for a monodisperse spherical cage structure. The aggregates appeared essentially complete within 60 min, with typical yields around 20% for both cross-linked TRAP cages (Fig. 1b). No free cysteine was detected after the reaction. Further analysis of the obtained TRAP cages using size exclusion chromatography coupled with right-angle and low-angle light scattering (SEC-RALS/LALS) showed an apparent average molecular mass of both particles as 2.2 MDa; A 24-ring organization was suggested.

The detailed structures for both TRAP-cage ^DTME and TRAP-cage ^BMH were determined using cryo-EM single particle reconstruction. Electron density maps at 4.7 Å and 4.9 Å resolution have been obtained for both types of cages. These revealed that each structure is composed of 24 TRAP rings organized into two chiral forms, similar to that seen for the TRAP cage ^Au(I) (Fig. 1e). . The TRAP ring model was refined to the map and produced a good fit. A more rigorous examination at the ring-ring interface found two substantial electron densities bridging two adjacent subunits (Fig. 1e), likely corresponding to a bismaleimide crosslinker. The crosslinker appears to be curved into a horseshoe shape between the cysteine residues of opposing subunits.

Both TRAP-caged ^DTME and TRAP-caged ^BMH exhibited similarly high stability in response to elevated temperatures, chaotropic agents, and surfactants. Specifically, they exhibited significant morphological changes after 10 min incubation at 75°C, pH in the range 2-11, up to 4 M GndHCl, up to at least 7 M urea, and 7% SDS. It was not presented. However, the TRAP cage ^DTME easily disassembles upon the addition of the reducing agents tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) (Fig. 2a,c). In contrast, TRAP cage ^BMH was unaffected (Fig. 2b,c). High stability at high temperatures, over a large pH range, and in the presence of high concentrations of chahortopic agents and surfactants was also observed for both TRAP-caged ^DTME (Fig. 2d–g) and TRAP-caged ^BMH . Ta. DTT-dependent disassembly was further investigated at the single cage level in real time using high-speed atomic force microscopy (HSAFM, Figure 2l). This indicated that TRAP-caged ^BMHs were resistant to disassembly in the presence of DTT. In contrast, TRAP cage ^DTME under the same conditions disassembles easily with discrete patches of TRAP subunits appearing to "peel off" from the cage surface, and the structure is disassembled approximately 3 min after the first ring detachment. This eventually led to a total opening (Fig. 2d). Such a gradual disassembly process of TRAP-caged ^DTME is in sharp contrast to TRAP-caged ^Au(I) , which shows a more coordinated disassembly over a much shorter time scale.

To efficiently track cage stability against various thiol-containing reagents, spectroscopic methods are employed to develop real-time monitoring of bulk cage disassembly in solution. This system encapsulates two fluorescent proteins, mOrange2 and mCherry, which act as Förster Resonance Energy Transfer (FRET) donors and acceptors, respectively. These fluorescent proteins were genetically fused to the TRAP N-terminus facing inside the cage in an associated structure. To avoid steric hindrance during the cage formation process, they were co-produced with unmodified TRAP to form a "patchwork" ring.

The resulting TRAP ring was then assembled into a cage structure using either Au(I) or DTME (Fig. 3a). After purification using size exclusion chromatography, the isolated particles were analyzed by native PAGE (Fig. 3b) and TEM imaging (Fig. 3c), which revealed guest encapsulation in the lumen of the monodisperse sphere cage. confirmed. The presence of both proteins in the confined volume of the TRAP cage lumen should enable efficient FRET (22, 23). Indeed, the normalized fluorescence spectra of TRAP-cage ^Au(I) co-packaged with both guests and TRAP-cage ^DTME are significantly different from the corresponding controls containing cages encapsulating only mOrange2 or mCherry and mixed in solution. Compared to the sample, it showed approximately 1.5 times higher signal in mCherry emission at 610 nm (Fig. 4a,b). However, addition of DTT, which induces disassembly for both types of cages, led to FRET cancellation observed in the resulting spectra, very similar to the control samples (Fig. 4a,b). These results demonstrated efficient energy transfer between encapsulated guests and their cancellation by release, indicating that the FRET cage is suitable for monitoring the TRAP cage disassembly process.

We next measured the disassembly kinetics for both TRAP-caged ^Au(I) and TRAP-caged ^DTME upon addition of DTT. Changes in the fluorescence intensity ratio at 568/610 nm were utilized as an indicator of the time-dependent disassembly process (Fig. 4c). For both cages, the increase in fluorescence reached a plateau approximately 5 minutes after addition of DTT, indicating complete cage disassembly and guest release. However, the mechanism of this process appeared to differ depending on cage type. In contrast to TRAP-cage ^Au(I) , TRAP-cage ^DTME exhibited an S-shaped disassembly behavior indicating multiple steps involved in the guest release procedure. These results were in good agreement with the coordinated versus stepwise disassembly of TRAP-cage ^Au(I) or TRAP-cage ^DTME , respectively, observed by HSAFM.

Protein cages that can carry cargo and disassemble in the presence of reducing agents have the potential for intracellular delivery. The ideal nano-vehicles used for this purpose should remain associated under extracellular conditions, but disassemble and release their cargo when exposed to intracellular conditions. We assessed this possibility by monitoring cage disassembly kinetics in the presence of cysteine and glutathione, employed as model thiols. A time-dependent increase in FRET cancellation was observed for TRAP cage ^Au(I) upon addition of cysteine, reaching a plateau after approximately 15 min, indicating complete cage disassembly (Fig. 4d, filled circles). In contrast, TRAP cage ^DTME containing the same FRET pair showed almost no fluorescence change, suggesting that the cage does not disassemble under these conditions (Fig. 4d, red circles). This stability towards cysteine is likely due to the slow kinetics of disulfide cleavage catalyzed by this free thiol, in contrast to the possibly much faster ligand exchange to the TRAP cage ^Au(I) . Nevertheless, TRAP-caged ^DTME can still be disassembled by using higher concentrations of free thiol compounds: significantly more disassembled when using 50 mM reduced glutathione compared to TRAP-caged ^Au(I). Disassembly of the TRAP cage ^DTME and release of cargo from it was observed, albeit at a slow disassembly rate (Fig. 4e, red and black circles).

According to embodiments, the cages described herein can be used as a medicine. This includes administering a cage as described herein to a patient or in treating a patient, such as including a cage as described herein for use in treating a disease in a patient. It can be. This may be a cage designed to carry cargo and disintegrate in the presence of a reducing agent, especially for intracellular delivery. These cages may be administered with or in the presence of pharmaceutically acceptable carriers, adjuvants, or excipients. A cage for medicinal use or for treating a patient would be beneficial to said patient. For example, as drug delivery systems (DDS) for active molecules (biological macromolecules such as RNA, DNA, peptides, and proteins, among others), biological macromolecules are often facilitated by conditions such as those found in vivo. They provide benefits because they are not destroyed or digested. Biological macromolecules are too large to diffuse through the holes in TRAP, a large protein, and the TRAP cage can continue to undergo significant changes without disrupting the overall structure. This means that it can be modified to capture a therapeutic cargo and simultaneously modified externally to target a therapeutic target. Programmable linkers can be used that cleave at desired circumstances that correlate with arrival at the site of action. For example, light can be shined onto the target site to cleave an open photocleavable TRAP cage. If the TRAP cage is permeated into the cell, the one held together by the reducible linker will spontaneously open and release the cargo as the cell's cytoplasm becomes highly reduced. The cage can also be used in conjunction with or in acting as a vaccine, where antigens (i.e. peptides) predicted to stimulate a T cell response are captured inside the TRAP cage and then targeted to T cells. followed by an induced aperture.

FIG. 2 is a diagram of molecular crosslinker-mediated TRAP cage formation. a, Schematic diagram of the crosslinking reaction using dithiobismaleimidoethane (DTME) or bismaleimidohexane (BMH). The TRAP (K35C/R64S) rings shown on the left, with cysteine represented as a circle on the outside, are covalently connected to each other via a reaction between the cysteine and a bismaleimide compound (detailed chemistry below). (line above the first arrow), forming a cage-like structure. Addition of dithiothreitol (DTT) results in DTME-mediated cage disassembly (TRAP cage ^DTME , top) but has no effect on cages assembled with BMH (TRAP cage ^BMH , bottom). b, Native PAGE gel for TRAP cage ^DTME and TRAP cage ^BMH formations with black arrowheads marking the height of the cages formed. c, Size exclusion chromatography profile for purified TRAP-cage ^DTME (light gray line) and TRAP-cage ^BMH (gray line). The TRAP cage ^Au(I) profile (black line) serves as a control. mAu, milli-absorbance unit. d, Transmission electron microscopy (TEM) images for TRAP-cage ^DTME (right) and TRAP-cage ^BMH (left). Scale bar, 50nm. e, Cryo-electron microscopy density map for left-handed (top) and right-handed (bottom) forms of TRAP cage ^DTME refined to 4.7 Å and 4.9 Å resolution, respectively. Inset shows an amplified image at the ring-ring interface with the fitted crosslinker model highlighted for DTME (middle) and BMH (right) shown in side view (top) and top view (bottom). ing. Figure 2: Stability of cross-linked TRAP cages. Redox responsiveness. a, b, Native PAGE analysis for TRAP-caged ^DTME (a) and TRAP-caged ^BMH (b) in the presence of DTT and tris(2-carboxyethyl)phosphine (TCEP). The TRAP cage appears as a prominent band running between 1048 and 1236 kDa. C=TRAP cage ^DTME (a) and TRAP cage ^BMH (b). M = molecular weight marker. c, TEM images showing TRAP cage ^DTME after treatment with 0.1 mM (left) and 1 mM (middle) TCEP, and TRAP cage ^BMH after treatment with 10 mM TCEP (right). Scale bar, 50nm. d–g, Stability of TRAP cage ^DTME . d, Native PAGE showing thermal stability of TRAP cage ^DTME over the indicated incubation times and temperatures. The image below the gel shows that the TRAP cage retains its structure after 10 min incubation at 95 °C, scale bar, 100 nm. e, Native PAGE showing the effect of pH on stability: The cage is stable from pH 3 to 11 using native PAGE. The image below the gel is a TEM image of the sample after incubation at the indicated pH. Scale bar, 100 nm. f, Structure of TRAP cage ^DTME assessed using native PAGE, showing that it is unaffected in the presence of guanidine hydrochloride (GdnHCl), urea, and g, SDS over the range tested. . TEM image (bottom) was acquired after incubation of the cage with 4M GndHCl, scale bar, 100 nm. h-k, TRAP-cage ^BMH stability, as for TRAP-cage ^DTME results (panels d-g), except that TRAP-cage ^BMH was used. Black arrowheads indicate the position of the intact TRAP cage on the gel. l, Frame from HSAFM video showing the effect of 4mM DTT addition to TRAP cage ^DTME . The time after addition of DTT is as indicated. FIG. 2 is a diagram showing a TRAP cage carrying a FRET pair. a, Schematic representation of a TRAP cage loaded with fluorescent proteins. Patchwork TRAP rings fused at the N-terminus with either mCherry (black cylinder) or mOrange2 (gray cylinder) mixed together with either DTME or triphenylphosphine monosulfate (TPPMS)-Au(I)-Cl did. b, Native PAGE showing the fluorescent properties of purified TRAP cages associated with fluorescent cargo. Gels were visualized using InstantBlue protein stain (right) and fluorescence using excitation at 532 nm and emission at 610 nm (left). Note that the exact location of the prominent band corresponding to the TRAP cage, running approximately 1028-1236 kDa, varies slightly according to the presence/absence of cargo and the nature of the crosslinker used. c, TEM images of an empty (left) TRAP cage and one filled with fluorescent protein (right) assembled using either Au(I) (top) or DTME (bottom). Scale bar, 50nm. FIG. 3 is a diagram of guest release. a, b, Normalized results of TRAP-caged ^Au(I) (a) and TRAP-caged ^DTME (b) carrying both mOrange2 and mCherry upon excitation at 510 nm before and after addition of 10 mM DTT. emission spectrum. mOrange2 emission peak at 568 nm, mCherry emission peak at 610 nm. Additional lines show spectra of cages carrying only mOrange2 or mCherry proteins mixed together just before measurement, in the absence or presence of DTT, respectively. c, d, e, TRAP-cage ^Au(I) (black circles) and TRAP-cage ^DTME after addition of 10mM DTT (c), 2.5mM cysteine (Cys) (d), or 50mM glutathione (GSH) (e). (gray circle) time-dependent disassembly. 100% leakage represents the highest donor strength, with 10 min of 10 mM DTT treatment after each experiment. FIG. 3 is a diagram of TRAP cages using various metal linkers. a. Native PAGE analysis for TRAP cages assembled with Ag(I) or Cd(II); b. TEM image of TRAP cage ^Ag(I) ; c. TEM image of TRAP cage ^Cd(I) ; d. Native PAGE analysis for TRAP cages associated with Co(II) or Zn(II); e. TEM image of TRAP cage ^Co(II) ; f. TEM image of TRAP cage ^Zn(II) ; a. and d. Samples were run on 3-12% native Bis-Tris acrylamide gels. Protein bands were visualized by Coomassie blue staining. Scale bar, 100 nm for all TEM images. Native PAGE of template reaction; M-marker; R-TRAP ring; C-TRAP cage control; C+T-TRAP cage + 10mM TCEP, showing cage disorganization due to the presence of Au(I); D=TRAP ring. +DBX-no cage association; DC-TRAP cage + DBX; DCT-TRAP cage + DBX + 10mM TCEP-cage still present, indicating successful exchange reaction; b. Structure of 1,3-dibromoxylene; c. DLS showing the approximate size of the DBX TRAP cage, 24 nm; d. TEM image of purified DBX TRAP cages after treatment with 10 mM DTT, scale bar, 100 nm; e. Cryo-EM structures for the leavo (left) and dextro (right) structures of the DBX cross-linked cage; f. Wireframe models of Au(I) inductive cage (left) and DBX cage (right) zoomed in on ring-to-ring connectivity; g. Structure of 1,2-bisbromomethyl-3-nitrobenzene (BBN); h. SDS PAGE before and after “template reaction” with BBN; M-marker, R-TRAP ring, C-Au(I)-inducible TRAP cage, BBN-TRAP cage after mixing with BBN-additional The appearance of a band indicates the presence of covalently bound TRAP dimers; i. TEM images of purified BBN TRAP cages, scale bars, 100 nm (left), 50 nm (right), j. Dependence of photocleavage on the presence of various quencher (DTT) concentrations after 10 minutes of UV irradiation, k. j. Showing various time points after the start of UV irradiation in the presence of 10 mM DTT. Native PAGE; for both gels: M-marker, C-TRAP cage, C+ or CDTT-crosslinked TRAP cage with reducing agent added to indicate its resistance to reducing conditions. FIG. 3 is a cryo-EM density map showing the structure of a TRAP cage fabricated using TRAP S33C/R64S, resulting in a 20-ring cage. From left to right: image focused on the 4x hole; image focused on the bowtie hole; perspective image; scale bar -5 nm.

Techniques adopted in the realization of the invention: Transmission Electron Microscopy (TEM)
Samples are typically diluted to a final protein concentration of 0.025 mg/ml, centrifuged briefly in a tabletop centrifuge, and the supernatant transferred to a hydrophilized carbon-coated copper grid (STEM Co.), negative staining using 4% phosphotungstic acid, pH 8, and visualization using a JEOL JEM-1230 80 kV instrument.

Native PAGE
Samples were run on 3-12% native Bis-Tris gels according to the manufacturer's recommendations (Life Technologies). Samples were mixed with 4x native PAGE sample buffer (200mM BisTris, pH 7.2, 40% w/v glycerol, 0.015% w/v bromophenol blue). NativeMark unstained protein standards (Life Technologies) were used as a qualitative guide to the molecular weight of migrated bands. When Blue Native PAGE was performed, protein bands were visualized according to the manufacturer's protocol (Life Technologies), otherwise using the InstantBlue™ protein stain (Expedeon).

Protein Expression and Purification A typical purification involves E. coli transformed with the pET21b plasmid carrying the TRAP (K35C/R64S) gene. E. coli BL21 (DE3) cells (Novagen) were grown in 3 L of LB medium with 100 μg/ml ampicillin at 37° C. with shaking until OD ₆₀₀ =0.6 and induced with 0.5 mM IPTG; It was then further shaken for 4 hours. Cells were harvested by centrifugation and pellets were kept at -80°C until use. Cells were lysed by sonication in 50 ml of 50 mM Tris-HCl, pH 7.9, 50 mM NaCl at 4°C in the presence of proteinase inhibitors (Thermo Scientific) and the presence or absence of 2mM DTT; The lysate was centrifuged at 66,063g for 0.5 h at 4°C. The supernatant fraction was heated at 70°C for 10 min, cooled to 4°C, and centrifuged again at 66,063 g for 0.5 h at 4°C. The supernatant fraction was purified on an AKTA purifier (GE Healthcare Life Sciences) using a 4 x 5 ml HiTrap QFF column with binding in 50mM Tris-HCl, pH 7.9, 0.05M NaCl, ±2mM DTT buffer. Purified by ion exchange chromatography, eluting with a 0.05-1M NaCl gradient. Fractions containing TRAP protein were pooled and concentrated using an Amicon Ultra 10kDa MWCO centrifugal filter unit (Millipore), and the samples were loaded with HiLoad in 50mM Tris-HCl, pH 7.9, 0.15M NaCl at room temperature. Size exclusion chromatography was performed on a 16/60 Superdex 200 column. Protein concentration was calculated using the BCA protein assay kit (Pierce Biotechnology).

Cage Stability The stability of TRAP cages to chemicals and heat was tested using methods similar to those previously described ^. All agents used in the assay (DTT, TCEP, SDS, Gdn-HCl, and urea) were reconstituted in PBS pH 7.4 and mixed with the TRAP cage samples overnight at room temperature. Thermal stability checks were performed by heating the samples at varying temperatures for 10 minutes. The samples were then subjected to native PAGE. These experiments were repeated twice, each giving uniform results.

Cryo EM
Glassy ice preparation was performed using 4 μL of protein sample at approximately 1 mg/mL in PBS. After blotting the samples onto EM grids (Quantifoil 1.2/1.3, Cu, 300 mesh), they were subjected to the following parameters: Blot power = 0, Blot time = 4 seconds, Wait time = 0 seconds, Drain time Frozen by submerging in liquid ethane using a FEI Vitrobot using =0 seconds. Photomicrographs were collected using a FEI TitanKrios cryomicroscope operating at 300 kV and a Falcon III camera at 75k magnification. 4942 and 10169 micrographs were collected for TRAP cage ^DTME and TRAP cage ^BMH , respectively. All micrographs were motion corrected using ^MotionCorr26 and CTF estimation was performed using ^CTFFIND47 . Particles were selected and extracted using cryoSPARC v2.12.4 ⁸ , first in manual mode (approximately 4000 particles) and then in automatic mode, with the initial 2D class serving as a template. The extracted particles were again 2D classified to select the best particles for the subsequent reconstruction step. 3D reconstruction was performed by Heterogenous Refinement protocol using EMD-4443 and EMD-4444 (TRAP cage ^Au(I) ) as search models.

Example 1. TRAP Cage ^DTME and TRAP Cage ^BMH Preparation Molecular Cloning For all cloning steps, E. E. coli NEB 5 alpha strain was used. Plasmid sequences were confirmed by Sanger sequencing performed by Eurofins. Tetracycline-inducible protein expression vectors were constructed by subcloning the gene segment encoding TRAP(K35C) into pACTet_H-mCherry or pACTet_H-mOrange. The gene for TRAP (K35C) was amplified by PCR using pET21b_TRAP-K35C as template and oligonucleotides FW_XhoI_TRAP and RV_MluI_TRAP as primers (see Table 3). The amplified PCR product was used directly as a template for a second PCR, which introduced the linker gene segment using FW_BsrGI_tev and RV_MluI_TRAP oligonucleotides as primers. The PCR products were cloned into pACTet_H-mCherry or pACTet_H-mOrange through the BsrGI and MluI sites to give pACTet_H-mCherry-TRAP-K35C and pACTet_H-mOrange-tev-TRAP-K35C. Here, ^only the single point mutation K35C, which is essential for subunit linkage, was introduced into the TRAP sequence, since the previously used R64S was the only one important to avoid gold nanoparticle binding.

Protein Expression and Purification TRAP protein is described in A. D. Essentially the same protocol as previously described in Malay et al., “An ultra-stable gold-coordinated protein cage displaying reversible assembly,” Nature 569, 438-442 (2019). but 2mM DTT was kept in buffer for the initial purification step to avoid undesired cysteine oxidation. To produce a patchwork TRAP ring, E. E. coli strain BL21 (DE3) cells were co-transformed with either pACTet_H-mOrange-TRAPK35C or pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C (see Table 4 in Materials and Methods). Cells were grown in 100 ml LB medium supplemented with ampicillin and chloramphenicol at 37°C to OD ₆₀₀ =0.5-0.7. At this point, protein expression was determined by the addition of 0.2 mM IPTG and 10 ng/ml tetracycline for pACTet_H-mCherry-TRAP-K35C or 30 ng/ml tetracycline for pACTet_H-mOrange-TRAP-K35C. was followed by cell culture for 20 hours at 25°C. Cells were then harvested by centrifugation at 5,000 xg for 10 minutes. Cell pellets were stored at -80°C until purification. They were then added to 40 ml lysis buffer (50 mM sodium phosphate buffer, 600 mM NaCl, 10 mM imidazole, pH 7.4) supplemented with a spatula tip of DNase I and lysozyme, 1 tablet of protease inhibitor cocktail, and 2 mM DTT. ) and stirred at room temperature for 30 minutes. Samples were then sonicated and clarified by centrifugation at 10,000 x g and 4°C for 20 min. The supernatant was then incubated for 20 minutes with 4 ml Ni-NTA resin pre-equilibrated in lysis buffer in a gravity flow column. The resin was then washed over 10 column volumes in lysis buffer containing 20 and 40 mM imidazole. His-tagged proteins were eluted using 5ml of 50mM sodium phosphate buffer (pH 7.4) containing 500mM imidazole. The protein sample was then filtered using an Amicon Ultra-15 centrifugal filter unit (50k molecular weight cutoff (MWCO)) (Merck Millipore) in 5mM ethylenediamine, hereinafter referred to as 2x PBS-E. Buffer exchange was performed into 2x phosphate buffered saline (PBS) supplemented with tetraacetic acid (EDTA). The protein was then subjected to size exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare) at a flow rate of 0.8 ml/min. The main peaks exhibiting absorption at 548 nm or 587 nm were pooled and concentrated using an Amicon Ultra-15 (50k MWCO). Protein purity was checked by SDD-PAGE, and protein concentration was determined using extinction coefficients: ε _{mCherry 587} = 72000 M ⁻¹ cm ⁻¹ , ε _{mOrange 548} = 58000 M ⁻¹ cm ⁻¹ , ε _{TRAP 280} = 8250 M ⁻¹ cm ⁻¹ was determined by the absorbance measured using a UV-1900 UV-Vis spectrophotometer (Shimadzu Corporation) (http://expasy.org/tools/protparam.html). Proteins were stored at 4°C until use.

Free Thiol Concentration Measurement Free thiol concentration in either TRAP-caged ^DTME and TRAP-caged ^BMH was assessed using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent according to the manufacturer's protocol. . Both samples were concentrated to 0.3mM using an Amicon Ultra-4 centrifugal filter unit (100k MWCO). Absorbance at 412 nm was measured using a Spectramax 190 UV/VIS plate reader (Molecular Devices). The concentration of free thiols in the samples was calculated from the molar extinction coefficient of 2-nitro-5-thiobenzoic acid (14150 M ⁻¹ cm ⁻¹ ) and was undetectable for TRAP cage ^DTME and TRAP cage ^BMH .

Cage Association and Purification For crosslinker-induced cage association, TRAP(K35C/R64S) (100-500 μM) in 2× PBS-E was mixed with a 5-fold molar excess of either DTME or BMH and incubated at room temperature. The mixture was stirred for 1 hour. The final DMSO concentration in the solution was kept no greater than 12.5%. After the reaction, the insoluble fraction, probably caused by the low solubility of the crosslinker in aqueous solution, was removed by centrifugation at 12,000×g for 5 min. The supernatant was then purified by size exclusion chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare) at a flow rate of 0.5 ml/min on an AKTA purifier (GE Healthcare). Fractions containing cross-linked TRAP cages were pooled and concentrated using an Amicon Ultra-4 (100k MWCO) centrifugal filter unit. The typical yield of cross-linked TRAP cages obtained was approximately 20%. Formation and purification of gold(I)-induced TRAP cages ^was performed as previously described. described for both cross-linked and gold(I)-derived cages using an additional Ni-NTA purification step before size exclusion chromatography to purify the sample from only partially associated cages. Cage formation with the fusion proteins was performed using the same protocol as (His-tagged mCherry and mOrange2, unprotected inside the cage, binds to the Ni-NTA column). Protein concentrations and ratios of encapsulated guests were estimated using absorbance ratios at 280/548 nm or 280/587 nm. The attenuation coefficients used in the calculation were ε _{mCherry 587} =72000M ⁻¹ cm ⁻¹ , ε _{mOrange2 548} =58000M ⁻¹ cm ⁻¹ , and ε _{TRAP 280} =8250M ⁻¹ cm ⁻¹ . Due to the spectral overlap between mCherry and mOrange2, the absorbance ratio at 548/587 nm of mCherry without fusion to TRAP was used to properly calculate the concentration of both encapsulated guests. The mCherry extinction coefficient was also estimated at 548 nm (ε _{mCherry 548} =42538M ⁻¹ cm ⁻¹ ). Similarly, the extinction coefficients of mCherry and mOrange2 at 280 nm were determined experimentally as ε _{mCherry 280} =56744M ⁻¹ cm ⁻¹ and ε _{mOrange2 280} =52200M ⁻¹ cm ⁻¹ , respectively.

Example 2. Confirmation of TRAP cage structure using cryo-EM The detailed structure for both TRAP cage ^DTME and TRAP cage ^BMH was determined using cryo-EM single particle reconstruction. It has acquired electron density maps at 4.7 Å and 4.9 Å resolution for both types of cages. These revealed that each structure is composed of 24 TRAP rings organized into two chiral forms, similar to that seen for the TRAP cage ^Au(I) . The TRAP ring model was refined to the map and produced a good fit. A more rigorous examination of the ring-ring interface found two substantial electron densities bridging two adjacent subunits, likely corresponding to a bismaleimide crosslinker. The crosslinker appears to be curved into a horseshoe shape between the cysteine residues of opposing subunits (Figure 1).

Example 3. Specific cleavage characteristics of molecular cross-linkers used in TRAP cages (Figures 2-4)
Both TRAP-caged ^DTME and TRAP-caged ^BMH exhibited similarly high stability in response to elevated temperatures, chaotropic agents, and surfactants. Specifically, they exhibited significant morphological changes after 10 min incubation at 75°C, pH in the range 2-11, up to 4 M GndHCl, up to at least 7 M urea, and 7% SDS. It was not presented. However, TRAP cage ^DTME is easily disassembled upon the addition of reducing agents tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT). In contrast, TRAP cage ^BMH was unaffected. DTT-dependent disassembly was further investigated at the single-cage level in real time using high-speed atomic force microscopy (HSAFM). This indicated that TRAP-caged ^BMHs were resistant to disassembly in the presence of DTT. In contrast, TRAP cage ^DTME under the same conditions disassembles easily with discrete patches of TRAP subunits appearing to "peel off" from the cage surface, and the structure is disassembled approximately 3 min after the first ring detachment. Eventually led to the opening of the whole thing. Such a gradual disassembly process of TRAP-caged ^DTME is in sharp contrast to TRAP-caged ^Au(I) , which shows a more coordinated disassembly over a much shorter time scale. The association of TRAP cage ^DTME with the FRET protein pari crog (Figure 3) also allows the disassembly in the presence of reducing agents to be characterized kinetically (Figure 4).

Example 4. Cage-associated protein expression and purification using various metals TRAP (K35C/R64S) protein was expressed and purified as previously described. TRAP(S33H/K35C) and TRAP(S33H/K35H) proteins were expressed and purified following the same protocol as TRAP(K35C/R64S), but all buffers had a pH of 8.5.

Protein concentration was determined by measuring absorbance at 280 nm.

Cage Association with Different Metals Purified TRAP variants (0.1 mM final concentration for monomeric subunits) and TRAP monomer:metal ion ratios of 4:1 to 2:1 in appropriate buffers. Salts of related metals: AgNO3 in 50mM Tris, pH _7.9 , 0.15M NaNO3; Cd( _NO3 ) _{2 in} 50mM Tris, pH 7.9, 0.15M NaCl; 50mM HEPES, pH 7.9, 0.15M Formation of TRAP cages was performed by mixing _CoCl2 or _ZnCl2 in NaCl. Reactions were typically incubated for 3 days at room temperature. TRAP cage formation was confirmed using native PAGE and TEM. Any precipitated material was removed by centrifugation at 12,045g for 5 minutes.

Gold propellant TRAP cage [TRAP (K35C/R64S) + Au (I)]
The tryptophan RNA binding attenuation protein double mutant TRAP (K35C/R64S) can associate into hollow sphere structures by reaction with monovalent gold ions. Cryo-EM single particle reconstruction shows that the resulting 22 nm cage is composed of 24 cyclic 11-mer subunits via linear sulfur-Au(I)-sulfur bridges between opposing cysteines. revealed.

Silver and cadmium propelled TRAP cage [TRAP(K35C/R64S) + Ag(I) or Cd(II)]
Cage formation can be promoted by other metals other than Au(I), namely Hg(II), Ag(I), Cd(II), and metal-promoted cage formation is shown in water-soluble compounds with a preferred two-ligand linear configuration. This suggests that d10 metal ions are required.

Ag(I)-TRAP cages form and remain stable only in the absence of chloride ions.

TRAP (K35C/R64S) cages created by addition of Ag(I) or Cd(II) showed bands on native PAGE with similar mobilities as Au(I)-mediated TRAP cages (Fig. 5a). Cage formation was further confirmed by negative stain transmission electron microscopy (TEM), which showed spherical hollow structures with a diameter of 22-24 nm (Figures 5b and 5c). These results suggested that silver and cadmium propelled cages could form structures with similar morphology to Au(I)-TRAP cages.

Cobalt and zinc propelled TRAP cage [TRAP(S33H/K35C) or TRAP(S33H/K35H) + Co(II) or Zn(II)]
The TRAP metal binding site has been reengineered to target metal ions with a tetrahedral coordination preference. Based on the crystal structure, histidine or cysteine and histidine pairs were introduced at the i and i+2 positions of the β-sheet motif at the periphery of the TRAP ring, yielding TRAP(S33H/K35C) and TRAP(S33H/K35H). , whereby each individual monomeric unit provides two ligands that link the divalent metal. These variants assembled into cage structures upon addition of Zn(II) and Co(II).

Native electrophoresis revealed that TRAP with both Zn(II) and Co(II) (S33H/K35H) migrated similarly to the Au(I)-mediated TRAP cage (Fig. 5d). The formation of a cage with a diameter around 22 nm was confirmed by negative stain transmission electron microscopy (TEM), suggesting the similarity of this structure to the Au(I)-TRAP cage (Figures 5e and 5f).

Example 5. TRAP cage materials and methods assembled using photocleavable crosslinkers:
Gold-inducible TRAP cages were prepared as previously described (Malay et al., Nature, 2019, herein incorporated by reference). 1,3-Dibromoxylene and 1,3-bisbromomethyl-4-nitrobenzene were purchased from commercial suppliers and dissolved in N,N-dimethylformamide (DMF). A 2 molar excess (relative to TRAP monomer) of either crosslinker was added to the freshly purified solution in 50 mM sodium phosphate buffer, pH 7.4, containing 5 mM EDTA with stirring for 1 h at room temperature. mixed with gold-induced TRAP cages. 10 mM dithiothreitol (DTT) was then added to the reaction to capture Au(I). The sample was then purified by size exclusion chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare).

1,3-bisbromomethyl-4-nitron - Light-induced disassembly of benzene TRAP cages was tested. Cage morphology and crosslinker cleavage process were monitored using dynamic light scattering (DLS) on a Zetasizer (Malvern), SDS, native PAGE, and TEM.

Results In a first attempt, we performed a similar method of cage association as we did for the BMH/DTME crosslinker (simply removing the TRAP ring and excess crosslinker). (mixing) and failed, probably due to the difficulty of controlling the correct orientation of the rings in the structure.

We used pre-assembly instead of just a ring, which allows the dibromo crosslinker to exchange gold atoms without changing the orientation of the ring in the cage, which we term a "templated reaction." We have found a novel method to overcome this challenge using an Au(I)-inducible TRAP cage. We further used 1,3-dibromoxylene (DBX) crosslinker (Fig. 6b) as a basis for optimization of this method. Gold-induced TRAP cages possessing Au(I) as an inter-ring linker tend to disassemble in the presence of reducing conditions (Fig. 6a, lane CT). We used this property to check whether DBX is incorporated into the structure of the TRAP cage as a result of a "templated reaction" due to its lack of disassembly properties in the same conditions. Indeed, after mixing DBX and Au(I)-induced TRAP cages and further purification, the TRAP cages become resistant to disassembly in reducing conditions, providing the presence of different types of linkages between the rings. We further characterized the structure of the obtained DBX TRAP cage by DLS method (Fig. 6c), which showed that the size of the DBX TRAP cage is approximately 24 nm with high monodispersity. showed that. The DBX TRAP cages were 2 nm larger than the Au(I)-induced TRAP cages, with the maintenance of a cage-like structure that could be observed in TEM, also suggesting the presence of cross-linkers in the structure, broadening their size (Fig. 6d). Finally, we solved the structure of the DBX TRAP cage demonstrating the presence of the DBX crosslinker between the rings. The cryo-EM structure of the resulting cage (Fig. 6e) revealed other interesting properties of the aggregate. Indeed, as in the case of the Au(I)-induced cage, we can distinguish between two chiral forms of the cage (leavo and dextro), which are due to the chiral nature of the gold-induced cage. It wasn't a surprise. A notable difference between the template cage (gold-induced) and the resulting one (DBX-containing) is the number of connections between the TRAP rings. For the Au(I)-induced cage, 120 connections were identified (-S-Au-S- bridge), while for the DBX cage, the number of connections drops to half that number. The 60 linker molecules and the same overall geometry (based on a modified cube) impose a slightly different TRAP ring orientation. Wireframe models of the Au(I)-induced cage and the DBX cage show the difference between the TRAP ring orientation and the transition from end-to-end (Au(I)) to apex-to-apex (DBX) (Fig. 6f).

Obtaining good results with the basic dibromo bridge of the TRAP cage, we developed DBX, which is very similar in structure but is sensitive to UV (365 nm) light due to the presence of the nitro group. It was decided to replace the photolabile crosslinker 1,2-dibromo-3-nitro-benzene, which can be cleaved after irradiation (Figure 6g). We optimized the conditions for the "template reaction" with the BBN crosslinker, which was found to be identical to the previously used DBX. SDS PAGE showed the clear appearance of TRAP dimers after the “templating reaction” (Fig. 6h), providing the presence of covalent bonds, indicating that the BBN cross-linker binds TRAP in a manner similar to the previously used DBX. It was suggested that it would be incorporated into the cage structure. TEM confirmed the presence of monodisperse TRAP cages with a diameter of approximately 24 nm (Fig. 6i).

To further investigate the potential of the acquired photolabile TRAP cages, we tested their ability for disassembly under UV light. Such reactions depend on the presence of a quencher, which does not make the reaction reversible. We tested various concentrations of quencher (DTT) and showed such dependence also in the case of BBN TRAP cages. BBN TRAP cages were successfully disassembled under UV light and in the presence of 10 mM DTT (Fig. 6j). We also tested how long it takes to completely disassemble a BBN TRAP cage. Native PAGE showed a gradual disappearance of the cage band in time, and complete disassembly was estimated to occur within approximately 2 minutes from the start of UV irradiation (Fig. 6k).

Throughout the description and claims of this specification, the words "comprise" and "containing" and variations thereof mean "including, but not limited to," and which , is not intended to (does not exclude) other parts, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, when the indefinite article is used, the specification is to be understood to contemplate the plurality as well as the singularity, unless the context requires otherwise.

A feature, integer, feature, compound, chemical moiety, or group that is described in conjunction with a particular aspect, embodiment, or example of the invention is not described herein unless it is incompatible with the use thereof. It should be understood that the invention is applicable to any other aspect, embodiment, or example of the invention. All of the features disclosed in this specification (including any appended claims, abstract, and drawings) and/or all of the steps of any method or process so disclosed may be incorporated herein by reference. At least some of such features and/or steps may be combined in any combination except those combinations that are mutually exclusive. The invention is not limited to the details of any previously described embodiments. The invention extends to any novel single or novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings). It extends to any novel single or novel combination of steps in any method or process.

Example 6. TRAP Cage Made from 20 Circles Materials and Methods The TRAP protein is as described above, except that the expression plasmid encodes a TRAP protein with the mutation S33C instead of K35C (also has the mutation R64S). It was expressed as follows. Incubation with the source of Au(I) was additionally similar to that described above. Subsequent purification of the resulting formed TRAP cages was similar to that described above. Determination of the structure of the resulting TRAP cage was performed using cryo-EM similar to that described above.

Results Structural analysis of the assembled cage (Figure 7) shows that it consists of 20 TRAPS33C/R64S rings connected with a cross-linking density reminiscent of that seen in the case of cages seen when using the TRAPK35C mutant. revealed that it will be configured. This suggests that, despite the lower resolution in this case, the connections between adjacent rings are similar to previous TRAP cages with Au(I) ions acting as bridges between two opposing Cys residues. Gives you confidence that the gold staples are the same as seen.

Claims (40)

An artificial TRAP cage containing a selected number of TRAP rings held in place by a molecular cross-linker, the cross-linkers being selected for their specific characteristics, whereby the cage is adapted to specific conditions. an artificial TRAP cage that is programmable to open or remain closed on demand. The specific cleavage characteristics of molecular crosslinkers are
(i) reduction-resistant/insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions;
(ii) a reduction-responsive/sensitive molecular cross-linker, whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular cross-linker, whereby the cage opens upon exposure to light. The cage of claim 1 selected from the group. 3. A cage according to claim 1 or 2, wherein the crosslinking agent is a homobifunctional molecular moiety and its derivatives. 4. A cage according to any one of claims 1 to 3, which is resistant or insensitive to reducing conditions. Cage according to claim 4, wherein the crosslinking agent is bismaleimidohexane (BMH) or bis-bromoxylene. 4. A cage according to any one of claims 1 to 3, which is responsive or sensitive to reducing conditions. 7. The cage of claim 6, wherein the crosslinking agent is dithiobismaleimidoethane (DTME). 4. A cage according to any one of claims 1 to 3, which is photoactivatable. The crosslinking agents include bis-halomethylbenzene, as well as 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3- 9. The cage of claim 8, which is a derivative thereof including bis-bromomethyl-4,6-dinitro-benzene (BDNB). 10. A cage according to claim 8 or 9, wherein the crosslinking agent is photolabile upon exposure to UV light. Cage according to any one of claims 1 to 10, wherein the number of TRAP rings in the TRAP cage is from 6 to 60. Cage according to claim 11, wherein the number of TRAP rings in the TRAP cage is 12, 20 or 24, preferably 24. 13. A cage according to any one of claims 1 to 12, comprising a mixture of different programmable crosslinking agents. 14. A cage according to any one of claims 1 to 13, encapsulating cargo that can be programmed to deliver cargo at specific times and at desired locations. 15. The artificial TRAP cage protein is modified to contain any one or more mutations selected from the group comprising K35C, R64S and K35C/R64S. cage. 16. A cage according to any one of claims 1 to 15, which is stable at elevated temperatures, stable at non-neutral pH and/or stable at chaotropic agents. An artificial TRAP cage comprising a selected number of TRAP rings held in place by at least one metal crosslinker, the metals being Ag(I), Cd(II), Zn(II), and Co. An artificial TRAP cage selected from the group comprising (II). Artificial TRAP cage proteins include K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/K35C 18. The cage of claim 17, wherein the cage is modified to include any one or more of the mutations selected from the group. 19. A cage according to claim 17 or claim 18, wherein the cross-linking agent comprises cadmium and preferably the artificial TRAP cage protein is modified to contain the K35C/R64S mutation. 19. The cage of claim 17 or claim 18, wherein the cross-linking agent comprises silver and the artificial TRAP cage protein is modified to contain the K35C/R64S mutation. 19. The cage of claim 17 or claim 18, wherein the cross-linking agent comprises cobalt and the artificial TRAP cage protein is modified to contain a S33H/K35C or S33H/K35H mutation. 19. The cage of claim 17 or claim 18, wherein the cross-linking agent comprises zinc and the artificial TRAP cage protein is modified to contain a S33H/K35C or S33H/K35H mutation. 23. A cage according to any one of claims 17 to 22, wherein the number of TRAP rings in the TRAP cage is from 6 to 60. 24. A cage according to claim 23, wherein the number of TRAP rings in the TRAP cage is 12, 20 or 24, preferably 24. 25. A cage according to any one of claims 17 to 24, comprising a mixture of different programmable crosslinking agents. 26. A cage according to any one of claims 17 to 25, encapsulating cargo that can be programmed to deliver cargo at specific times and at desired locations. 27. A cage according to any preceding claim, wherein the cage is approximately spherical in shape. 28. Use of a cage according to any one of claims 1 to 27 in the delivery of cargo in a controlled period and to a desired location. 28. Use of a cage according to any one of claims 1 to 27 as a medicament. 28. A method of treating a patient, comprising administering to the patient a cage according to any one of claims 1 to 27. 28. A cage according to any one of claims 1 to 27 for use in treating a disease in a patient. Use of any one or more of groups, including homobifunctional molecular moieties, bis-halomethylbenzene, and derivatives thereof, as programmable cross-linking agents in the construction of programmable TRAP cages. Use of any one or more of the metals Ag(I), Cd(II), Zn(II), and Co(II), and derivatives thereof, as crosslinking agents in the construction of TRAP cages. (i) obtaining the TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system;
(ii) conjugation of the TRAP ring unit via at least one free thiol linkage with a programmable molecular crosslinker, where the crosslinker is selected for its specific characteristics;
(iii) formation of the TRAP cage; and (iv) purification and isolation of the TRAP cage. Programmable crosslinker
(i) a reduction-resistant/insensitive linker, whereby the cage remains closed under reducing conditions;
(ii) a reduction-responsive/sensitive linker, whereby the cage opens under reducing conditions; and (iii) a light-activatable linker, whereby the cage opens upon exposure to light. 35. The method of claim 34. 36. A method according to claim 34 or 35, wherein step (ii) comprises conjugation with a mixture of different programmable crosslinking agents. 37. A TRAP cage produced by the method of any one of claims 34-36. (i) obtaining the TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system;
(ii) conjugation of the TRAP ring unit via at least one free thiol linkage with a metal crosslinker, where the crosslinker is selected for its specific characteristics;
(iii) formation of the TRAP cage; and (iv) purification and isolation of the TRAP cage, wherein the metal is Ag(I), Cd(II), Zn(II). , and Co(II). A TRAP cage produced by the method of claim 38. A mutation selected from the group comprising K35C, K35H, R64S, K35C/R64S, K35H/R64S, S33C, S33H, S33C/R64S, S33H/R64S, S33C/K35H, S33H/K35H, S33C/K35C, and S33H/K35C An artificial TRAP cage comprising a protein modified to include any one or more of: